Clostridium difficile (C. difficile) Toxins A and B are responsible for C. difficile-associated disease (CDAD), which manifests itself as nosocomial diarrhea and pseudomembranous colitis (Kuijper et al., Clinical Microbiology and Infection 12(Suppl. 6):2-18, 2006; Drudy et al., International Journal of Infectious Diseases 11(1):5-10, 2007; Warny et al., Lancet 366(9491):1079-1084, 2005; Dove et al., Infection and Immunity 58(2):480-488, 1990; Barroso et al., Nucleic Acids Research 18(13):4004, 1990).
Toxins A and B are encoded by two separate but closely linked (and highly homologous) genes. Toxins A and B are produced simultaneously in C. difficile strain VPI 10463 (ATCC 43255), and the ratio of the produced toxins is usually 3:1, respectively (Karlsson et al., Microbiology 145:1683-1693, 1999). The toxins begin to be formed during the exponential growth phase, and are usually released from the bacteria between 36 and 72 hours of culture. Toxins present within the bacteria can be released earlier by sonication or by use of a French pressure cell.
Treatment of the toxins with formaldehyde results in the corresponding Toxoids A and B, which are completely inactivated and retain at least partial immunogenicity (Torres et al., Infection and Immunity 63(12):4619-4627, 1995). It has been shown that vaccination employing both toxoids is effective in hamsters, healthy adults, and patients with recurrent CDAD (Torres et al., Infection and Immunity 63(12):4619-4627, 1995; Kotloff et al., Infection and Immunity 69(2):988-995, 2001; Sougioultzis et al., Gastroenterology 128(3):764-770, 2005; Tones et al., Vaccine Research 5(3):149-162, 1996). Additionally, the administration of both free and aluminum salt (adjuvant) bound toxoids leads to appropriate immune responses (Torres et al., Vaccine Research 5(3):149-162, 1996; Giannasca et al., Infection and Immunity 67(2):527-538, 1999).
The administration of both toxoids simultaneously is more effective than administration of the individual proteins alone (Kim et al., Infection and Immunity 55(12):2984-2992, 1987). A toxoid composition found effective in inducing protective immune responses against toxin A and toxin B in patients with recurrent CDAD included both toxoids, at a ratio of 1.5:1, A:B (Sougioultzis et al., Gastroenterology 128(3):764-770, 2005).
Both the A and B toxoids are thus candidates for vaccine development. Greater production efficiency of Toxins A and B is desired to facilitate vaccine production.